Vascular calcification occurs when hydroxyapatite (HA) is deposited in cardiovascular tissues such as arteries and heart valves. HA can be a significant risk factor in the pathogenesis of cardiovascular disease and has been associated with myocardial infarction and coronary death (Detrano R C, Doherty T M, Davies M J, Stary H C 2000 “Predicting coronary events with coronary calcium: pathophysiologic and clinical problems.” Curr Probl Cardiol 25:374-402). The mechanisms of pathological vascular calcification are believed to be similar to normal embryonic bone formation (Doherty T M, Uzui H, Fitzpatrick L A, Tripathi P V, Dunstan C R, Asotra K, Rajavashisth T B 2002 “Rationale for the role of osteoclast-like cells in arterial calcification.” Faseb J 16:577-582). Studies have demonstrated an association between low bone mass and an increased risk of cardiovascular disease (von der Recke P, Hansen M A, Hassager C 1999 “The association between low bone mass at the menopause and cardiovascular mortality.” Am J Med 106:273-278).
The link between cardiovascular disease and bone formation has been verified in vivo. Matrix Gla Protein (MGP)-deficient mice (Mgp−/−), for example, display an osteopenic bone phenotype with arterial calcification (Speer M Y, McKee M D, Guldberg R E, Liaw L, Yang H Y, Tung E, Karsenty G, Giachelli C M 2002 “Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla protein-deficient mice: evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo.” J Exp Med 196:1047-1055). Mutations affecting the osteoclastic lineage, such as in osteoprotegerin (OPG) knockout mice, which have an osteoporotic phenotype, are also associated with arterial calcification (Bucay N, Sarosi I, Dunstan C R, Morony S, Tarpley J, Capparelli C, Scully S, Tan H L, Xu W, Lacey D L, Boyle W J, Simonet W S 1998 osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 12:1260-1268). In addition, osteopontin (OPN), a mineralization inhibitor, is known to have dual roles in bone and heart (Steitz S A, Speer M Y, McKee M D, Liaw L, Almeida M, Yang H, Giachelli C M 2002 “Osteopontin inhibits mineral deposition and promotes regression of ectopic calcification.” Am J Pathol 161:2035-2046). OPN is expressed in osteoblasts as well as in activated inflammatory cells in injured arteries and appears to play a protective role against arterial calcification, as OPN null mice are compromised when responding to cardiovascular challenge (Myers D L, Harmon K J, Lindner V, Liaw L 2003 Alterations of arterial physiology in osteopontin-null mice. Arterioscler Thromb Vasc Biol 23:1021-1028).
These observations support the contention that bone mineralization and arterial calcification share similar underlying pathologies. Furthermore, mice lacking NPP1 (Enpp1−/−), a major generator of the calcification inhibitor inorganic pyrophosphate (PPi), spontaneously develop articular cartilage, perispinal and aortic calcification at a young age (Okawa A, Nakamura I, Goto S, Moriya H, Nakamura Y, Ikegawa S1998 “Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine.” Nat Genet. 19:271-273). These mice share similar phenotypic features with a human disease, idiopathic infantile arterial calcification (IIAC) (Rutsch F, Vaingankar S, Johnson K, Goldfine I, Maddux B, Schauerte P, Kalhoff H, Sano K, Boisvert W A, “Superti-Furga A, Terkeltaub R 2001 PC-1 nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification.” Am J Pathol 158:543-554; Rutsch F, Ruf N, Vaingankar S, Toliat M R, Suk A, Hohne W, Schauer G, Lehmann M, Roscioli T, Schnabel D, Epplen J T, Knisely A, Superti-Furga A, McGill J, Filippone M, Sinaiko A R, Vallance H, Hinrichs B, Smith W, Ferre M, Terkeltaub R, Nurnberg P 2003 “Mutations in ENPP1 are associated with ‘idiopathic’ infantile arterial calcification.” Nat Genet. 34:379-381). Moreover, in another mouse model with depressed extracellular PPi (ePPi) levels, due to defective transport function of the transmembrane protein ANK (ank/ank mutant mice), soft tissue ossification is found, similarly to that in Enpp1−/− mice (Ho A M, Johnson M D, Kingsley D M 2000 “Role of the mouse ank gene in control of tissue calcification and arthritis.” Science 289:265-270-13; Harmey D, Hessle L, Narisawa S, Johnson K, Terkeltaub R, Milián J L 2004 “Concerted regulation of inorganic pyrophosphate and osteopontin by Akp2, Enpp1 and Ank. An integrated model of the pathogenesis of mineralization disorders.” Am J Pathol 164: 1199-1209; Johnson K, Polewski M, van Etten D, Terkeltaub R 2005 “Chondrogenesis mediated by PPi depletion promotes spontaneous aortic calcification in NPP1−/− mice.” Arterioscler Thromb Vasc Biol 25:686-691).
Alkaline phosphatases (E.C.3.1.3.1) (APs) are dimeric enzymes present in most organisms (Milián J L 2006 “Mammalian alkaline phosphatases. From biology to applications in medicine and biotechnology.” Wiley-VCH Verlag GmbH & Co, Weinheim, Germany pp. 1-322). They catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate (Pi) and alcohol. In humans, three of the four isozymes are tissue-specific, i.e., the intestinal (IAP), placental (PLAP), and germ cell (GCAP) APs, while the fourth AP is tissue-nonspecific (TNAP) and is expressed in bone, liver and kidney.
Recent studies have provided compelling evidence that a major role for TNAP in bone tissue is to hydrolyze ePPi to avoid accumulation of this mineralization inhibitor, thus ensuring normal bone mineralization (Johnson K A, Hessle L, Wennberg C, Mauro S, Narisawa S, Goding J, Sano K, Milián J L, Terkeltaub R 2000 “Tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) act as selective and mutual antagonists of mineralizing activity by murine osteoblasts.” Am J Phys Regulatory and Integrative Physiology 279: R1365-1377-17; Hessle L, Johnson K A, Anderson H C, Narisawa S, Sali A, Goding J W, Terkeltaub R, Milián J L 2002 “Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.” Proc Natl Acad Sci USA 99:9445-9449; Johnson K, Goding J, Van Etten D, Sali A, Hu S I, Farley D, Krug H, Hessle L, Milián J L, Terkeltaub R 2003 “Linked deficiencies in extracellular PP(i) and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression.” J Bone Min Res 18:994-1004). Normalization of ePPi levels in NPP1 null and ANK-deficient mice improves their soft-tissue ossification abnormalities (Johnson K A, Hessle L, Wennberg C, Mauro S, Narisawa S, Goding J, Sano K, Milián J L, Terkeltaub R 2000 “Tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) act as selective and mutual antagonists of mineralizing activity by murine osteoblasts.” Am J Phys Regulatory and Integrative Physiology 279: R1365-1377, 16; Hessle L, Johnson K A, Anderson H C, Narisawa S, Sali A, Goding J W, Terkeltaub R, Milián J L 2002 “Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization.” Proc Natl Acad Sci USA 99:9445-9449). Crossbreeding either the Enpp1−/− or the ank/ank mice to mice deficient in TNAP (Akp2−/−) mice normalizes ePPi levels and induces a secondary up-regulation of OPN levels (Johnson K, Goding J, Van Etten D, Sali A, Hu S I, Farley D, Krug H, Hessle L, Milián J L, Terkeltaub R 2003 “Linked deficiencies in extracellular PP(i) and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression.” J Bone Min Res 18:994-1004).
Importantly, these studies have indicated that TNAP may be a useful therapeutic target for the treatment of diseases such as ankylosis and osteoarthritis, but also arterial calcification. Indeed, substantial evidence points to the presence of TNAP-rich vesicles at sites of mineralization in human arteries. The presence of TNAP-enriched matrix vesicles (MVs) in human atherosclerotic lesions suggests an active role in the promotion of the accompanying vascular calcification (Hsu H H, Camacho N P 1999 “Isolation of calcifiable versicles from human atherosclerotic aortas.” Atherosclerosis 143:353-362; Hui M, Li S Q, Holmyard D, Cheng P 1997 “Stable transfection of nonosteogenic cell lines with tissue nonspecific alkaline phosphatase enhances mineral deposition both in the presence and absence of beta-glycerophosphate: possible role for alkaline phosphatase in pathological mineralization.” Calcified Tissue International 60:467-72; Hui M, Tenenbaum H C 1998 “New face of an old enzyme: alkaline phosphatase may contribute to human tissue aging by inducing tissue hardening and calcification.” Anatomical Record 253:91-94. Tanimura A, McGregor D H, Anderson H C 1986 “Calcification in atherosclerosis. I. Human studies.” J Exp Pathol 2:261-273. Tanimura A, McGregor D H, Anderson H C 1986 “Calcification in atherosclerosis. II. Animal studies.” J Exp Pathol 2:275-297). Increased expression of TNAP accelerates calcification by bovine vascular smooth muscle cells (VSMCs)(Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M, Morii H 1995 Beta-glycerophosphate accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 15:2003-2009) and macrophages may induce a calcifying phenotype in human VSMCs by activating TNAP in the presence of IFNγ and 1,25 (OH)2D3(Shioi A, Katagi M, Okuno Y, Mori K, Jono S, Koyama H, Nishizawa Y 2002 “Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages.” Circ Res 91:9-16). Calcification of rat aorta in culture and of human valve interstitial cells has been shown to be dependent on TNAP activity (Lomashvili K, Cobbs S, Hennigar R, Hardcastle K, O'Neill W C 2004 “Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin.” J. Am. Soc. Nephrol. 15: 1392-1401; Mathieu P, Voisine P, Pepin A, Shetty R, Savard N, Dagenais F 2005 “Calcification of human valve interstitial cells is dependent on alkaline phosphatase activity.” J Heart Valve Disease 14:353-357).